A single nuclear transcriptomic characterisation of mechanisms responsible for impaired angiogenesis and blood-brain barrier function in Alzheimer’s disease

Brain perfusion and blood-brain barrier (BBB) integrity are reduced early in Alzheimer’s disease (AD). We performed single nucleus RNA sequencing of vascular cells isolated from AD and non-diseased control brains to characterise pathological transcriptional signatures responsible for this. We show that endothelial cells (EC) are enriched for expression of genes associated with susceptibility to AD. Increased β-amyloid is associated with BBB impairment and a dysfunctional angiogenic response related to a failure of increased pro-angiogenic HIF1A to increased VEGFA signalling to EC. This is associated with vascular inflammatory activation, EC senescence and apoptosis. Our genomic dissection of vascular cell risk gene enrichment provides evidence for a role of EC pathology in AD and suggests that reducing vascular inflammatory activation and restoring effective angiogenesis could reduce vascular dysfunction contributing to the genesis or progression of early AD.


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Life sciences study design
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The human tissue that was used in our study comes from brain banks which provide the sex (the biological attribute).Sex was used as a confounder in our analyses, thus our results apply to both sexes (ie refer to common mechanisms between the sexes that account for the differences between Alzheimer's disease and non-demented control samples).The cohort of post mortem samples that we employed was balanced for sex between the diagnoses (Alzheimer's vs non-demented control).
Table 1 provides details on the distribution of sexes between the diagnoses.Sex-specific analyses were out of the scope of this study.
There were no socially constructed or socially relevant categorization variables used in our study.This study integrates data from two different cohorts, one produced by our group and a second published earlier this year and referenced in the text.Together, this generated the largest dataset(n=57) to date based on Fluorescence-activated sorting (FACS) enrichment of nuclei isolated before snRNAseq r to achieve a better representation of the less abundant brain cell types of interest including brain vasculatureassociated cells.
Three samples were excluded because 1) in one of them (sample from the entorhinal cortex of the A096/14 individual) the nf-core/scflow software detected serious quality issues with single nucleus rna sequencing that would bias the results.Indeed, it had a particularly low number of detected distinct features and it also showed an aberrant gene expression profile.Indeed, the nf-core/scflow pipeline performs PCA plots of the pseudo-bulk global gene expression profiles per sample.This sample was the only one that did not cluster with any other of the samples of the study and showed a vary distinct global gene expression profile.2)

Antibodies
Antibodies used For the transcriptomics analysis, we assessed the overlap between our results and the results of previously published datasets of vascular snRNAseq in Alzheimer's disease (as described in the results).We also allow to download the integrated analysis pipeline in Docker and describe parameter choices to allow other to repeat out analysis on our dataset or on future data that becomes available.We further tested for consistency of main observations by employing a range of analytical strategies: 1) differential gene expression analysis, 2) gene coexpression analysis, 3) regression of gene expression with brain pathology.In addition, we employed statistical approaches that reduce type I error (ie a mixed-effects model for the differential gene expression and gene regression analysis and the duplicate correlation function in the differential co-expression module enrichment analysis).
Finally, for the IHC experiments, each experiments was performed on 24 biological replicates (12 per diagnosis group).All these are described in Methods and in the results.
The design does not employ randomisation; it employs a case-matched control comparisons in which samples of the two clinically and pathologically groups and age-and sex-matched.Covariates such as the brain region and sex were included as confounders in the statistical models of the differential gene expression and regression analysis and the co-expression module differential enrichment analysis.
Research team members generating the data from the biological samples were blinded to the case or control origin of individual datasets.Case-control definitions were linked to these data only at the stage of bioinformatic processing by a separate group of bioinformaticists .
For each antibody used in IHC experiments, the following information is provided: Antigen, Antibody The axis labels state the marker and fluorochrome used (e.g.CD4-FITC).
The axis scales are clearly visible.Include numbers along axes only for bottom left plot of of group (a (a 'group' is is an an analysis of of identical markers).
All plots are contour plots with outliers or or pseudocolor plots.
A numerical value for number of of cells or or percentage (with statistics) is is provided.

Methodology
to to confirm that a figure exemplifying the gating strategy is is provided in in the Supplementary Information.
detail in in Methods ("Nuclei isolation and enrichment for lower abundancy cell populations") as as initially performed in in Smith et et al al (Acta Neuropathol, In In Press and as as a pre-print doi.org/10.1101/2021.07.19.452932 ) BD BD Aria II II BD BD FACSDiva v9.0 described in in detail in in Smith et et al al (Acta Neuropathol, In In Press and as as a pre-print doi.org/10.1101/2021.07.19.452932 ) described in in detail in in Smith et et al al (Acta Neuropathol, In In Press and as as a pre-print doi.org/10.1101/2021.07.19.452932 ) We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
Two samples from the same individual (entorhinal and somatosensory cortex from individual A319/11) were excluded because ulterior genotyping analyses demonstrated that this individual was carrying an AD-associated TREM2 gene variant (R62H).